Despite the availability of antibiotics, tuberculosis remains a major cause of death worldwide. Current drugs, while effective and inexpensive, are quite difficult to administer because of the very extended courses of treatment required for successful therapy. Current antibiotics require a substantial infrastructure to deliver and supervise therapy, an investment that is difficult in much of the world. Antibiotics that acted more rapidly could lead to much more effective cure and control of disease. We have previously defined the set of bacterial genes that are required for optimal in vitro growth of Mycobacterium tuberculosis. Here we propose to create strains that conditionally express genes that encode predicted secreted and cell wall proteins. This will enable us to probe their importance under various growth conditions, including those in which bacteria are growing rapidly and various models of nonreplicating persistence. In addition, we will use the same strains to identify genes which, when inactivated, are cleared rapidly in a mouse model of infection. Finally, we will use a genetic screen that has already enabled us to identify unexpected synergistic drug combinations to study a broad range of potential antituberculous compounds. Altogether, these results should allow more rational selection of targets for treatment of tuberculosis.